THE PRODUCTION OF INDOLE AUXINS AND THEIR EFFECTS ON GROWTH

OF FUNGUS AT DIFFERENT STAGES

A thesis submitted in part fulfilment

of the-requirements for the degree of

Master of Science

Shung-ko Ng

Division of Biology

Graduate School

The Chinese University of Hong Kong

June 1972

1Contents

Tables and illustrations iii

Introduction 1

Materials and methods 7

(1) Materials 7

(2) Culturing of the fungi 9

(3) Extraction of the indole compounds 12

(4) Paper chromatography and spot detection 14

(5) Determination of reference quantity of indole compounds 20

(6) Distingushing dead from living yeast cells 25

(7) Measuring of the cell number and cell size 28

(8) Measuring of gas consumption and yield 29

(9) Measuring of sugar (glucose) decreased 32

(10) Measuring of alcohol yield 36

Results 38

(1) The indole compounds produced by the fungi 38

(2) The effect of auxin IAA on the growth curve of yeast 41

(3) The measurements of gases during the growth stages of

yeast 50

(4) The sugar used during the growth stages of yeast 54

(5) The alcohol production at different stages of yeast

growth 56

(6) Other changes during the growth stages of yeast 59

Discussion 63

(1) Indole compounds production 63

(2) Physiology of auxin on yeast 64

2

(3) Suggestions for application 67

Summary 68

Acknowledgements 71

Literature cited 72

3Tables and illustrations

Figure 1. Set-up for paper-chromatography 15

Figure 2. Transmissible wave length by red cellophane 23

Figure 3. Set-up for measuring CO2 yield 30

Table 1. Detection of indole compounds on paper-chromatography 40

Figure 4. The control growth curve of Saccharomyces cerevisiae 42

Figure 5. The growth curve of Saccharomyces cerevisiae in

liquid medium containing 1 mg/l of IAA 44

Figure 6. The growth curve of Saccharomyces cerevisiae in

Jiquid medium containing 5 mg/l of IAA 45

Figure 7. The growth curve of Saccharomyces cerevisiae in

liquid medium containing 10 mg/l of IAA 46

Figure B. The change of concentration in exogenous indole

compounds at different stages of growth in

Saccharomyces cerevisiae 48

Figure 9. Carbon dioxide yield by Saccharomyces cerevisiae 51

Figure 10. The consumption of oxygen by different types of

cultures of Saccharom ces cerevisiae 53

Figure 11. Percentage of sugar (glucose) remainded in the

liquid medium55

Table 2. Quantity of alcohol yield during yeast growth and

the% by weight of production 57

Table 3. Cell size (u) of yeast at different time of growth 60

Table 4. Summary of data available at the present time 61

1

THE PRODUCTION OF INDOLE AUXINS AND THEIR EFFECTS ON GROWTH

OF FUNGUS AT DIFFERENT STAGES

Indole-3-acetic acid (IAA) has been known for many years

as a kind of plant growth substance. It was the glory of the

microbiologists that the production of this exciting, simple but

important organic compound of great influence to plant growth

and development was first discovered by Salkowski (1880) in the

common fungus, Rhizopus suuinus. Almost at the same time, Charles

0

and Francis Darwin (188) observed the curvature of young seedlings

to light. About 20 years later, Hopkins and Cole (1903) reported

that IAA was found in Escherichia coli (Migula) Castellani and

Chalmers. Then, Boysen-Jensen (1919) reported that the cause of

seedling curvature to light is material. Demonstration of the

extraction of auxin and its polar transport were succeeded

about 10 years later (Went, ',1926).

Reports on auxin physiology in fungus are comparatively

less than in higher plants. Furthermore the research were usually

associated with their hosts (Fenner Fate, 1947; Wolf, 1952;

Chandramohan Mahadevan, 1968) Extraction and identification

of IAA seemed to be the main interest in the past years (;Alolf,

1952; Gruen, 1959 a b, 1965; Epstein Miles, 1967; Norberg,

21968; Runkova, 1969)

Many other growth substances were also discovered in fungi,

such as gibberellins in Gibb'erella fujikuroi (Sawada) Wollenweber

(Yabuta et al, 1938), and kinetin isolated from yeast DNA (Miller

et al, 1955). Even the newly discovered growth substance,

ethylene, a kind of gaseous growth substance which is very impor-

tant in fruit repening, have. also been observed in the production

of Penicillium digitatum Saccardo (Jacobsen Wang, 1968).

Since 1880, many workers have been engaged in the field

of auxin physiology. Unfortunately, as it is said by Wightman

and Setterfield (1968),...... the unsolved problem of the primary

action of auxin in regulating growth is still remained undissolved

Perhaps the difficulty for this slow progress is due to the comple-

xity of physiology of higher plants and most of the investigators

were also concentrated to the problem of auxin transport (McCready,

1966; Goldsmith, 1966,'1968; Lyon, 1965 a b; Naqvi et al, 1965

a by 1966, 1967) and interaction of the plant growth substances

(Thimann, 1963; Masingale et al, 1968; Witham, 1968; Khan, 1968;

Lodhi et al, 1968). Besides the time required for a higher plant

to complete its growth is rather long. Therefore most of the

investigators cannot afford to spend such a long time in doing a

piece of work. Nevertheless efforts have been applied to the

hormone actions in rdlation to nucleic acid metabolism (Galston

3

& Purves, 1960; Letham, 1967, 1969; Haber et all 1969; Penner et

all 1969; Holm, 1970; Shih Rappaport, 1970; Walton et all 1970),

to protein and enzyme metabolism (Cherry, 1968; Galston et all 1968;

Glasziou et,al, 1968; M acLachlan et all 1968, Palmer, 1968; Abeles

Forrence, 1970), and to cell wall physiology with cell ultra-

structure (Cleland, 1968; Hall Ordin, 1968; Moore C Eisinger,

1968; Ray Ordin, 1968).

During the past decades, if micro-organisms were used in

the studies of auxin physiology, then it would have developed

and expanded more quickly and fruitfully. For. examples, enzymo-

logy was initiated and developed by the studies of the enxymes

produced in yeast; molecular biology, biological chemistry,

microbial genetics and molecular genetics were initiated by the

studies with Neurospora (Beadle Tatum, 1941); and photosynthesis

was enriched by the application of green alga Chorella (Bassham

Calvin, 1957).

Unlike other fields of plant physiology, plant hormones

.(plant growth substances, plant growth regulators) or simply called

auxins, were in fact first studied in micro-organisms. It might

be our human beings' blindness or rather practical minded, studies

of plant hormones in applied fields are much more than that in

pure sciences. So that the results :are not sufficient enough to

4develop such an interesting field.

IAA research in fungi were concentrated on the studies of

its production (Salkowski, 1880; Wolf, 1952; Gruen, 1959 a b,

1965; Batista et al, 1966; Epstein 'Miles, 1967; Chandramohan

M ahadevan, 1968; Norberg, 1968). Production of a sex hormone was

studied in Act bisexualis (Raper, 1951; McMorries, 1967).

Production of other plant hormones were also studies in fungus

(Yabuta et al, 1938; Miller et.al, 1955; Jacobsen Wang, 1968;

Norberg, 1968).

The studies of physiologicl effects of auxins on fungi

were not well established. The knowledge of-this field is limited.

The inhibition of fungus mycelial growth by IAA was first reported

by Hessayon (1952). There was no effect in spore germination of

Phycomyces blakesleeanus with the treatment of IAA and gibberellic

acid to spores or substrate (Hocking, 1967). Degradation and trans-

forming of indole compounds were demonstrated in Aspergillus niger

78 (Dvornikova et al, 1968 a b).

Exogenous application of IAA obtained different responses

from fungi. Strong dose of IAA inhibits fungal growth (Jerebzoff-

Quintin, 1967). Fusarium oxysoorum var. cubense is inhibited by

5

IAA application of normal dose (200 mg/1- 800 mg/1) wile

Fusarium vasinfectum is highly resistant to IAA application

(Hessayon, 1952). Recovery of inhibition was demonstrated by

application of phthalic acid and some its esters in Nectria

galligena Bres. (Jerebzoff-Quintin, 1967). Cell expansion

induced by IAA was observed in some homothallic strains, of

S accharomyces ellipsoideus and Saccharomyces cerevisiae Meyen ex

Hansen, but the application of trans-cinnamic acid, actinomycin

D, chloramphenicol and cycloheximide showed the anti-auxin

effects (Yanagishima, 1966; Yanagishi1na Shimoda, 1968;

Yangishima et al,. 1970).

In this historical review, we see in the past that the

studies of auxin effects on fungi were rather limited. Observa-

tions were concentrated to morphological changes.rather than

physiological effects. It is difficult to study the influence of

auxin during the processes*of fungal metabolism.; without some

analysis of the products it produced, the use of medium and the

consumption of oxygen.

Therefore the intention of this report is to see what is

actually going on in yeast under the effect of exogenous applica-

tion of IAA. To what degree does this chemical affect the metabolic

6activities of the yeast and what is the relationship between the

concentration of the indole compounds and the yeast growth. At

the same time, IAA in application to fermentation industries is

also considered in this research.

7Materials and Methods

(1) Materials

(a) The yeast

Saccharomyces cerevisiae Meyen ex Hansen is commonly known as

Baker's Yeast. It is widely used in bakery, brewery, distillery

and many other industries.

The strain in our lahoratory was obtained from Mr. S. M.

Sun of the University of Hong Kong. It was purified in our labo-

ratory by dilution method. It is believed to be arisen from a

single vegetative cell or a single budding group. The identity

of the genetical and physiological characters is maintained with

such treatment.

(b) The Aspergillus niger NAC2

Aspergillus niger V. Tieghem is a kind of common molds known as

black mold occurring-in both foodstuffs and clothings. We isolated

this mold in 1968 and put into our culture collections.

The mutant, A. niger NAC2, was isolated from bur culture

collection in 1969. It vas first appeared as a colony sector.

Purification was done by single hyphal culture.

8

The basic structures of this mutant are similar to that

of A. ni er (Tam, Ng Bau, 1968). The differences between them

are:

1) Standard growth rate in Czapek's Solution Agar

Wild type--3.5 cm in colony diameter of 10-day growth

Mutant--5.0 cm in colony diameter of 10-day growth

2) The head

Wild type--brownish black to black

Mutant--black

3) Abundance of head and the time of development

Wild type--early and abundant

Mutant--rare especially in the first 10 day

The mutant was chosen for experimental purposes simply

because of its character of late and rare head formation. This

character gives advantages in overcoming the contamination and

cleanliness of filtrates as well as the mycelial mats for extrac-

tion.

9(2) Culturing of the fungi

(a) Culturing of the yeast

The stock culture was maintained in test-tubes containing

medium modified by Kinoshita (1927) with the following compositions:

NH4NO3 1.00 g

K2HPO4 1.00 g

MgSO4.7H2O0.50 g

Glucose (Dextrosol)30.00 g

Agar (Sigma type IV) 20.00 g

Distilled waterto make up 1 liter

pH=4 (adjusted with 4 N HC1 before autoclaving

Inoculum was prepared by transferring a loopful of yeast

cells from the stock into an Erlenmeyer flask (150 ml) containing

50 ml of the above liquid medium (agar free). The flask was then

fixed in gyrotory waterbath shaker at 30+1°C and with about 200

rpm for 48 hours. The culture was in the logarithmic phase at

this time. The concentration of the cell was determined by using

a haemacytometer and diluted to the desired concentration with

liquid medium.

10About 1 cell for each ml was inoculated into a 250 ml

Erlenmeyer flasks containing 200 ml of the liquid medium.

Appropriated quantity of IAA was added accordingly. Then the

flasks were fixed in Gyrotory waterbath shaker at 30'1°C and with

approximately 200 rpm. Samples were taken for examination at time

designated.

(b) Culturing of the Aspergillus

The stock was maintained in test-tubes containing Czapek's

Solution Agar modified by Dox (1910) with the following compositions:

NaNO3 3.00 g

K2HPO41.00 g

MgSO4.7H20 0.50 g

KC1 0.50 g

FeS04.7H20 0.01 g

Glucose (Dextrosol) 30.00 g

Agar (Sigma type IV) 15.00 g

Distilled water to make up 1 liter

pH=4 (adjusted with 4 N HC1 before autoclaving)

The inocula were prepared by subculturing of the stock in

petri dishes containing the above medium. Colonies were cut into

small pieces of about 2 mm2 before the head developed.

11Bottles of 7 cm in diameter and 8.5 cm in height containing

Czapek's liquid medium (agar free) of about 1/3 of its height were

used for culturing. One piece of the inoculum was put into each

bottle and was grown in complete darkness for 10 days at room

temperature of 25-28°C. Head development hence would be the

least.

12

(3) Extraction of the Indole compounds

After an appropriate'.period of growth (3 days for yeast

and 10 days for Aspergillus), the fungi were separated

from their liquid media by suction filtering in Aspergillus and

by centrifugalization in yeast with a refrigerated centrifuge

(Model RC2-B Sorvall) at 5000 rpm-'for 20 'miss.

(a) Exogenous Indole-compounds

The liquid media were\first acidified with 4 N HC1 to a

pH of 3.5. About 1/4 of the volume of the liquid -medium of

peroxide free diethyl-ether was added into a separation funnel

containing the liquid medium. Then they were shaken thoroughly

and kept in refrigerator at 5°C. Shakings were made at time

.intervals for. better extraction. After 2 days, the ether layer

was drained into clean numbered bottles and stored in refrigerator.

The extracting procedures were repeated three times.

(b) Endogenous Indole-compounds

To extract the endogenous indole-compounds, the fungi were

first washed thrice with deionized distilled water. They were

separated as described before. Then they were dried by filter

13

paper and acidified with a few drops of 4 N hydrochloric ;acid.

The mycelial mats of Aspergillus were mashed with mortar and

pestle. About 4 times of their volumes of absolute ethyl

alcohol was added into the fungal materials. Then the whcle

thing was cooled in salt-ice and sonified thoroughly. After-

wards it was put into refrigerator at 5°C for extraction for

3 days. Separation of the alcoholic layer was made possible by

centrifugalization. These procedures were repeated for three

times.

(c) Condensation of the Extracts

For condensation of the extracts, Buchi Rotavapor was

used. The ether extracts were condensed to approximately 1/20

of its original volume by vacuum distillation at room temperature

of 28°C and stored in refrigerator with numbered clean bottles.

The alcoholic extracts were condensed by vacuum distillation at

30-40°C and stored as before.

34

(4) Paper Chromatography and Spot Detection

The methods described here were basically similar to that

of Stowe and Thimann's (1954). Sometimes methods described by

Sen and Leopold (1954) were also used in order to get a better

result.

(a) Paper chromatography

Paper chromatography was developed on Whatman's No. 1

filter papers of 3 X.35 or 12 X,35 cm in a Shadon Chroma jar.

Special modification of the jar with a movable glass rod was

made for more efficiency (Fig. 1). The movable glass rod enables

the pushing down of the chromatograph for•:develcpment without

opening of the jar. Hence the saturation of the solvent vapor

in the jar can be maintained. The solvent used was iso-propanol:

ammonia (28%):distilled water= 8:1:1 (v/v/v).

Before the development, the paper and tank were saturated

with solvent vapor by setting them overnight. Pushing of the glass

rod until the lower end of the paper dipped into the solvent. The

development of paper chromatography was then begun.

15Glass rod

(Movable)

Cork

Cover

Pad

Hook

Chromajar

Pape

Line of

Application

Solvent

Fig. 1. Set-up for paper-chromatography

16

(b) Reagents of Color-reaction for spot detection

(I) Ehrlich's Reagent:

p-Dimethylaminobenzaldehyde (p-DMAB) reagent or better

known as Ehrlich's reagent has been widely used in detection of

indole-compounds. In reaction with indole-compounds, it gives

stable pink, purple or blue colors except some hydroxyindolyl

acetic acid.

About a dozen modifications in composition of this

reagent have been developed by various authors. The following

two compositions were found in this experiment to be more

efficient and better fit to the purpose:

Jepson's (1960) Modification

p-DMAB

HCl (conc.)

Acetone

10.00 g

20.00 ml

80.00 ml

The paper strips were dipped into this reagent for spot

detection. Color spots were appeared immediately after the

treatment. The sensitivity of this modification was very high.

It is said that it is sensitive up to 0.05 pg of IAA.

17Prochazka et al's Modification

Solution 1

p-DMAB

HC1 (12 N)

2.00 g

100.00 ml

Solution 2

NaNO2

Distilled water

1.00 g

100.00 ml

The paper was sprayed homogenously with solution 1 and

after 2-3 minutes, sprayed with solution 2. Then the paper was

°dried in an oven at 50 C. Color spots were appeared.

(II) Salkowski Reagent:

Salkowski.Reagent or stated in other books as FeC13-HC104

reagent was also widely used by many workers. The method stated

here was the one. modified by Gordon. and Weber (1951). The

composition of this reagent was as follows:

FeCl 3 (0.05 M)

HC104 (5.0%)

Ethyl alcohol

2.00 ml

100.00 ml

102.00 ml

18

The reagent was sprayed homogenously onto the paper with

the greatest caution. Its sensitivity to IAA was 0.1,ug and to

other indole compounds ranging from 10 pg or less. The color and

the Rf of the spots were immediately recorded, otherwise it change

very quickly and the paper would disintegrate within a short time.

(III) Diazotized Sulfanilic Acid Reagent:

This reagent was prepared by Ames and Mitchell (1952) and

was used by Stowe and Thimann (1954). The preparation of this

reagent requires quick and skillful technique with patience. The

procedures were as follows:

Twenty-five ml of freshly prepared 5;/0 NaNO2 was slowly added

I

at 0°C to 5 ml of sulfanilic acid solution (0.9 g sulfanilic acid

and 9 ml conc. HC1, diluted to 100 ml with distilled water).

The dried chromatograms were sprayed lightly with this

reagent. Color spots were obtained. Then the papers were sprayed

with% Na2C03 while they were still damp for keeping their disin-

tegrating at the minimum.

This reagent reacts with most indole compounds and gives

characteristic colors.

19

(IV) Diazotized p-Nitroaniline Reagent:

This reagent was prepared by Bray, Thorpe and'White (1950)

and was introduced by Stowe and Thimann (1954). For detection

of color spots, freshly prepared reagent was preferred. The

preparation procedures were as follows:

Twenty-five ml of p-nitroaniline (0.3%) in HC1 (80%) was

mixed with 1.5 ml.of ice cooled sodium nitrite just before spraying.

The spraying procedures were similar to those in reagent

III. Characteristic colors were observed in reaction with indole

compounds.

20

(5) Determination of Reference Quantity of Indole Compounds:

The quantitative determination of indole compounds seemed

to be very difficult. Since the yield of indole compounds was very

low (in mg/1).' The loss of it in extraction and separation was

undoubtly happened. Several authors have attemped to do the quan-

titative analysis of different indole compounds produced by higher

plants (Stowe et al, 1968; Phelps Sequeira, 1968). Generally

reference quantitative analysis is preferred.

(a) Reference Quantitative Analysis of Exogenous Indole

Compounds

If white light is passed through a solution containing

colored compounds, certain wavelengths of light are selectively

absorbed. The resultant intensity of light is due to the trans-

mitted light. The convertion of a compound into a colored sub-

stance may be quantitatively determined by calorimetric method.

Colorimetric method for determination of indole compounds have

been employed in research work (Libbert et al, 1966).

In this. experiment for determination of reference

21quantitative analysis of exogenous indole compounds, a Bausch and

Lomb Spectronic 20 spectrophotometer was used. The indole compounds

were first converted into colored compounds with Ehrlich's Reagent.

Since most indole compounds react with Ehrlich's Reagent to yield

a purplish colored compounds, so the percentage of trans-

mittance measured by the colorimeter would indicate the nearest

indole compounds concentration.

Three ml of the cell free liquid medium was added into a

standard matched test tube. Three ml of Ehrlich's Reagent (Prochazha

solution 1) was added. The solutions were mixed well and incubated

in water bath at SO°C for half an hour. The test tube and the

solution was cooled in a cold water bath and the surface of

test tube was dried and cleaned with tissue paper. The percentage

of transmittance was measured with a colorimeter at 580 mi.

(b) Reference Quantitative Analysis of Endogenous Indole

Compounds:

Crude extract of known number of cells according to most

probable number counted by haemocytometer was slowly evaporated to

driness. The crystal obtained was dissolved with water into 100 ml.

A 2 ml unit was used for bioassay.

22

The bioassay methods were basically similar to that of

Sirois (1960). Avena seeds of uniformed size were submerged in

running water for 2-3 hrs. Then they were surface sterilized

with 0.05% HgCl2 for 15 mins. The seeds were placed at an angle

of 45° with the embryo down. A 2-ply tissue paper was supported

by glass rods fixed at about 1/2 inch from the bottom of a plastic

tray with the edges of the tissue paper in contact with distilled

water below. This device kept the tissue paper moist by capillary

action. The tray and seeds were incubated in humidity and tempera-

ture chamber at 20-22°C with relative humidity of 90% for 24 hrs

from a red light source supplied by a Philip's electric bulb of

12 W wrapped by four layers of red cellophane which had been tested

to have the properties of stopping the transmittarfce of light shorter

than 570 mi (Fig. 2), Then the light was turned off and temperature

of the chamber was kept at 26-28°C until most of the coleoptiles

reached the height of 20 mm.

Cutting of the coleoptiles was carried out with razor blades

mounted on a cutting apparatus in dark room lightened with a faint

green light (Bentley, 1950). Customarily the apical 3 mm of the

coleoptiles was discarded and the following 5 mm segments were

floated on sterilized distilled water for 3 hrs in de-IAA process.

The coleoptile segments were transferred in groups of 10 to each

23Fig. 2. Transmissible wave length by red cellophane

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5350 400 450 500 550 600 650 700 750

WAVELENGTH (MILLIMICRONS)

ABSORBANCE

24

of Jena petri dishes which contained 20 ml of buffer and 2 ml of

the test solution. The controlled dishes contained 20 ml of buffer

only. The buffer system was composed as follows:

K2HPO4 1.794 g

Citric acid.H20 1.019 g.

Glucose (Mallinckrodt AR) 10.000 g

Distilled water To make up 1 liter

pH= 5.8

The length of the coleoptile segments was measured under

binocular with 10-fold magnification after a 20 hrs incubation

period in the dark at 26 0C.

2

(6) Distinguishing Dead from Living Yeast Cells

Yeast unlike Escherichia coli and many other bacteria,

budding is essential. Each budding group may contain 2-8 cells

or more. Hence viable count With plating method may at least

evolves of 79% error and it is still not efficient if we use

the method of measuring by optical density. Moreover some

yeast and bacterial cells at the death phase may lose the ability

of dividing but themselves are still living (Knaysi,1935). In

order to overcome these difficulties, vital staining method is

preferred.

A number of methods were tried in this experiment including

methylene blue (Leifson, 1951; Leifson and Hugh, 1953); Loeffler's

methylene blue and a few modifications.. None of these gave good

results. Hsui and Fong (1957) have reported some work on this

subject in Acta Microbiologia Sinica. A quick and reliable method

was developed by modification of Knaysi's method (1935). The

preparation of the stain was as follows:

One ml of Leoffler's methylene blue and 1 ml of neutral

red (1%) were diluted with 10 ml of distilled water. A few

crystals of M gSO407H20 were added as mordant. A few drops of the

stain were spread thinnly over a clean slide. Then the stain was

26evaporated to driness on hot plate of 50°C.

A drop of yeast solution was added onto the surface of the

slide and covered with a clean cover glass. Examinations were carried

out under microscope with oil immersion lens. The dead cells showed

red color and the living ones showed no color and if any, very faint

blue. Application of MgSO4 as the mordant was the criterion for

success. This modification was based mainly upon its mechanism of

action.

Living bacteria and yeasts are generally negatively charged

(Potter, 1911; Clark, 1919, 1920, 1923-25). But dead cells are

comparatively more positive and inert (Herbert Hawk, 1966). Hence

Mg in the stain are first adsrobed onto the surface of living cells.

It serves as

(1) the prevention of the forced penetration of neutral

red, and

(2) displacement by the methylene blue

(1/2n Mg) (yeast cellsn)+ nMB+Cl

(nMB+) (yeast cells n)+ 1/2n MgCl 2

Yet the methylene blue is subjected to be reduced by enzyme

dehydrogenase (Thumberg, 1920)

27Methylene blueI

Leucomethylene blue

rEnzyme H2

LEnzyme

Hence living cells would give no color or very faint blue.

The dead cells are electrically more positive and inert.

The pH value of yeast cells approximately equals to 6 (Mahdihassan,

1930; Gutstsin,1932). The redox potential (Eh in mV) of neutral

red and methylene blue on pH 6 are -279 and +47 respectively.

Therefore penetration of neutral red would be dominant. Hence

the color of the dead cells is red.

28

(7) Measuring of the cell number and cell size

Measuring of the cell number was made possible by using

a Haemacytometer. The cell suspensions were diluted to an

appropriate concentration (1/20). Under light microscope, the

number of cells in a definite volume of cell suspension can be

read. Well developed buds were counted as single cells.

The measuring of cell size was made possible by using

occulometer. The length and the width of the cells were taken.

29

(8) Measuring of Gas Consumption and Yield

The 10-day growth of yeast in relation to CO2 yield was

measured in a set-up shown in Fig. 3. Clean 'Merk' brown-glass

bottles of 1 liter sized were used as fermentor. One liter of the

yeast solution medium (prepared as before) was put into the bottle

and sterlized in autoclave. One cell per ml of yeast cells were

inoculated. The bottles were put into a water bath of 30°C and the

set-up was jointed. Heating of the water bath was controlled by a

fish thermostat heater and its circulation by a stirrer. The mano-

meter consisted of a burette and a milk pint bottle. The joints

and connections were sealed with silicon grease so that the

diffusion of gas was checked. The fermentation set-up was heated

for 3 hrs before closing of three-way stopcork so that the system

was completely concealed.

The wax film at the top of the water in the burette was

used to prevent the dissolving of CO2 in water and the oil film

in milk pint bottle was used to prevent the evaporation of water

into the atmosphere. Leveling of water, surfaces in burette and

milk pint bottle gave the exact outer and inner pressure. Greatest

care has been taken to avoid broken of the wax film. Hence the

volume of gas changed could be accurately measured. Two replicates

of control and 3 replicates of Saccharomyces cerevisiae fermentation

experiment were set up, and opening of the three-way stopcork'was

30

Three way

stopcork

-Wax-film

Milk pintO1l-film

-Water

-Rubber tube

-Water

Burette

-Fermentation tank

Stirrer

-Water bath

-Thermostat heater

Fig. 3. Set-up for measuring CO2 yield

31made at irregular time intervals for diffusion of oxygen inside

the system. For further investigation, the three-way stopcork was

closed again for measuring the CO2 yield.

For measuring the oxygen consumption during the first stage

of yeast growth, Warburg apparatus was used. Both flask and mano-

meter.,.of the Warburg apparatus were calibrated with mercury so

that the exact volume can be calculated. Numbered flasks containing

medium only (control), medium+ yeast cells, medium+ IAA Cl mg/l)+

yeast cells, medium+ IAA (5 mg/1)+ yeast cells, and medium+ IAA

(10 mg/1)+ yeast cells were investigated. Known volume of saturated

KOH solution was added into the middle container of Warburg flasks

so that any trace amount of CO2 could be removed. In order to increase

the efficiency of KOH action, cylindrical stripe of filter paper was

put into the middle container for increasing the absorption surface

and prevention of the splashing, about of KOH solution. By subtracting

the volume decreased in the control flask the CO2 gas in the flask

was eliminated. Volume decreased should be equivalent tho oxygen

consummed.

For re-aeration of the flask, the gas supplied was intro-

duced by a fish pump. The gas from atmosphere was first passed

through the coil immerged in the water bath for warming up, then

to fused CaCl2 for drying, to fused NaOH for removing C02, to

filter for removing micro-organisms, to sterilzed water for moistening

and finally distributed to flasks. After each re-aeration process

of about 15 minutes, a heating up process of approximately 1/2 hr

was needed.

32(9) Measuring of Sugar (Glucose) decreased

Somogyi-Shaffer-Hartman's method was used for quantitative

analysis of glucose content in residue of fermentation medium

(Good et al, 1933).

The theory was as follows:

(a) Divalent copper ions can be reduced by reducing sugar

to form monovalent copper ions

Cureducing sugar

Cu

(b) Iodide, can be oxidized by iodates to form free iodine

in acidic solution

5KI+ KIO3+ 3H2SO4 3K2SO4+ 3H2O+ 3I2

(c) The monovalent copper ions can reduce free iodine

into iodide and itself is oxidized into divalent copper ions again

2Cu + I2 2Cu + 2I

(d) The oxalate contained in the reagent will form

complex with the divalent copper ions and hence prevent backward

reaction of (c).

33

(e) The yield of Cu+ in reaction (a) can be measured by

titration of remaining iodine with sodium thiosulfate

I2+ 2Na2S203 Na2S406+ 2NaI

Preparation of Somogyi-Shaffer-Hartman's Reagent

CuSO405H2O 5.00 g

Tartaric acid 7.50 g

Anhydrous sodium carbonate 40.00 g

Potassium iodate 0.70 g

Potassium oxalate18.40 g

Redistilled water to make up 1 liter

Preparation of 0.005N sodium thiosulfate (AR)

Dissolved 25 g-of sodium thiosulfate in 500 ml of

boiling redistilled water. Waited until cold and diluted to 1 liter.

Calbrate the concentration:

Carefully weighed 0.15 to 0..18 g of KIO3 and dissolved it

in 50 ml of redistilled water. Added 10 ml each of 15`/3 KI solution

and 6 N H2SO4. Waited for 3 minutes and diluted to 150 ml. Titrated

with the above prepared sodium thiosulfate solution until the color

changed to light golden yellow. Added 5 ml of 1% soluble starch

solution. Titrated until the blue color of starch disappeared.

Used blank titration with 10 ml of 191% KIO3 for correction,

34Calculation of the sodium thiosulfate solution

KI03 (g)Conc.

214.03

Corrected result X

6000

To obtain 0.005N sodium thiosulfate

0.005 N X 1000= Conc. X n (vol. in ml wanted)

0.005 N X 1000

nConc.

Procedures:

The residues were diluted to appropriated concentration

of 1 mg/5 ml. Five ml of Somogyi-Shaffer-Hartman's reagent was

added to the same flask. Covered the flask with a funnel and

heated in boiling water bath for 30 minutes. The flask was taken

out without shaking and cooled down to room temperature. Then

5 ml of 1 N H2504 was added and the flask was shaken until no

further air bubbles were observed. The free iodine was titrated

with 0.005 N sodium thiosulfate until the color changed to light

golden yellow. One ml of i/13 soluble starch solution was added

as indicator. Further titration was made until the starch solution

35losing its blue color.

Calculations were based on that 1 ml of 0.005 N sodium

thiosulfate solution is equivalent to 0.318 mg of Cu+. Hence

the quantity of Cu+ is

(Vol. of Na2S203 used in blank titration- Vol. of Na 2 S 2 0 3 used

in sample titration) X 0.318 mg

But the reduction of Cu++ to Cu+ by glucose is not in a

very precise ratio, so the quantity of glucose in residue could

be found by the table (Good et al, 1933).

36

(10) Measuring of Alcohol Yield

For measuring of the alcohol (ethanol) produced at different

stages of growth, acetylation method was employed. About 0.01-0.02

M of alcohol in the residue was added into an iodine bottle. The

content of alcohol in the residue was first titrated and the

concentration determined roughly. Then calculated precise volume

of the residue that might contain the above quantity and added into

the flask. Thirty ml of acetic anhydride-pyridine reagent was also

added together with 2 ml of 6 N H2s04. The glass-stopper was.

moistened with pyridine and loosely seated. 'Then the iodine bottle

was put in water bath with boiling water and heated for 2 hrs.

Cooled at room temperature for a few minutes then cooled in ice

bath with the stopper partly opened. Added in a few drops of mixed

indicator and titrated with 0.1 N alcoholic sodium hydroxide

solution,

Preparation of the reagents

(a) Mixed indicator: mix 1 part of 1% aqueous cresol

red with 3 parts of thymol blue (1%).

(b) 0.1 N standard alcoholic sodium hydroxide: mix

saturated aqueous sodium hydroxide with ethanol and titrated with

37primary standard 1 N sulfuric acid and diluted to the desired

concentration,

(c) Acetylating reagent: mix one part of acetic anhydride

with 3 parts of pyridine.

*The reagents (b) and (c) were prepared every day.

30Results

(1) The indole compounds produced by the fungi

Six indole compounds have been discovered in yeast. Two

indole compounds were observed in liquid medium extracts. The

first compound with Rf of .76, purple in color on Ehrlich's test,

blue on Salkowski, and orange on diazotized p-nitroaniline, was

undoubtly indoleacetonitrile (IAN). The second with Rf of .35,

purple in color on Ehrlich's test, rosy red on Salkowski, light

brown on diazotized sulfanilic acid, was significant enough to be

indole-3-acetic acid (IAA). There was quite often a spot with Rf

of .15 and yellow in color on Ehrlich's test. It was preliminarily

determined as citrulline (Table l).

Three kinds of indole compounds were surely present-in

yeast cell extracts. They were indole-acetonitrile (IAN), indole

aldehyde (IAH) and indole-3-acetic acid (IAA). Other three indole

compounds were present occasionally. The one appeared most often

was a compound with Rf of .95 and very sensitive to Ehrlich's test.

The other two did not appear so often. They were with Rf of .53

and .33 respectively, acid positive to Ehrlich's test (Table 1).

In the liquid medium extracts of Aspergillus nigerr NAC2,

there were three types of indole compounds discovered. The

39compounds detected were indole (IND), indole-3-acetic acid (IAA),

and tryptophan (TTP) (Table 1).

In the alcoholic extracts of Aspergillus nigrr NAC2,

mycelial mats gave quite different indole compounds in comparison

with its liquid medium extracts. Three types of indole compounds

were obtained. Only one was identified which was skatole (SKT)

with Rf of .92, ash blue in color on Ehrlich's test, greyish

brown on Salllowski, light-orange yellow on diazotized p-nitroaniline,

and light-yellow on diazotized sulfanilic acid. The other two

compounds have not been identified with the observed data. The

first compound possessed Rf of .05 with yellow in color on Ehrlich's

test, pale yellow on Salllowski, colorless on diazotized p-nitroaniline,

and light-brown to yellow on diazotized sulfanilic acid. The second

compound possessed Rf of .37 with yellow in color on Ehrlich's test,

light-yellow on Salkowski, colorless on diazotized p-nitroaniline

and light-brown by graduately changed to yellow on diazotized

sulfanilic acid (Table 1).

40Table 16 Detection of Indole Compounds on Paper Chromatography

Diazotized DiazotizedOrigion Rf Ehrlich Salkowski Compounds

p-nitroaniline sulfanilic acid

orange76 purple blueYeast liquid Indoleacetonitrile

35 light-brownpurple rosy redmedium yellowish orange Indoleacetic acid

15 yellowCitrulline?

Yeast cell 76 orangepurple blue Indoleacetonitrile

70 orangepurple rosy red orange Indo.lealdehyde

35 light-brownpurple rosy red yellowish orange Indoleacetic acid

purple to95

greenish

blue

53 purple

33 purple

A. niger NAC2 .80 light-brownpink rosy red light-brown Indole

Liguid medium 37 light-brownpurple rosy red yellowish orange Indoleacetic acid

20 purple yellowish orange yellow Tryptophan

A. niger NAC2 ash blue92 light-brown greyish brown light-yellow Skatole

mycelial mat 05 colorlessyellow orange-pinkpale-yellow

37 colorlessyellow 1ight-yellow light-brown to

yellow

41

(2) The effect of IAA on the growth of yeast

Like many other single celled micro-organisms, the growth of

yeast can be divided into several stages or phases. Customarily,

the division of the growth stages are in accordance to the cell

population and the ratib between living and dead cells. In a most

way

simple, the growth of micro-organisms can be divided into four phases;

viz. the lag phase, the logarithmic phase, the stationary phase,

and the death phase.

(a) The standard growth curve of yeast

Under the conditions described in this cultural study, the

lag phase and the logarithmic phase of yeast lasted about 48 hrs.

During the first 30 hrs, the culture remained quite clear and seemed

nothing had happened. All the cells in the culture were alive at

this time. A sudden increase of cell number was observed after 30

hrs and soon reached to its climax at the 48th hour. After the 48th

hour or so, the culture went into stationary phase. Dead cells was

observed at this phase. The cell number per ml was reached log. B.

The stationary phase lasted about 8 to 10. days. The death phase

began with a rapid fall in the number of living cells, but the number

of the cells (i.e. total cells) did not decrease. Occassionally,

broken down of cells were observed and a small reduction of cell

number was resulted (Fig. 4).

42

Fig. 4. The control. growth curve of Saccharomyces cerevisiae

The lag phase and the log. phase last about 48 hrs. After

these two phases, the culture goes to stationary phase which

lasts about 8 to 10 days. Unlike bacterial growth at stationary

phase, yeast does contain some dead cells in the culture, i. e.

not all the cells are alive. Only when culture shows a rapid

fall in the number of living cells, the culture then enters to

the death phase.

Total cells

Living cells

Time of growth in days

10

8

6

4

2

00 2 4 6 8 10 12 14 16

18

Log.

no.

of

cells

43

(b) The effect of IAA on the growth curve of yeast

The doses of IAA used in. this investigation were 1 mg/l,

5 mg/l, and 10 mg/l. The common effect on growth curve of yeast

by these dosages was the extension of the early stages, i. e.

the leg phase and the log. phase. The extension of these two

phases by effect of IAA seemed to be not in proportion to the

concentration of IAA applied. The time of-lag phase and log,

phase extension by -the dosages of 1 mg/l and 5 mg/l seemed to be

equal, both was 48 hrs in comparison with standard growth curve.

The time of extension in the culture of 10 mg/l of IAA was 96

hrs. (Figs 5, 6, and 7).

44

Fig. 5. The growth of Saccharomyces cerevisiae in liquid

medium containing 1 mg/l of IAA

Note the extention of the Lag phase and the Log. phase. In

comparason with the control-.culture, the extention of these

two phases was up to approximately 48 hrs.

Total cells

Living cells

Time of growth in days

0 2 4 6 8 10 12

10

8

6

4

2

0

Log.no.ofcells

45

Fig. 6. The growth curve of Saccharomyces cerevisiae in liquid

medium containing 5 mg/1 of IAA

The extension of lag phase and log. phase is also existed. But

by taking growth curve on account alone, the fungal response in

this concentration gives no difference with that of 1 mg/l of IAA.

Total cells

Living cells

Time of growth in days

0 2 4 6 8 10 12

10

8

6

4

2

0

Log.

no.

of

cells

46

Fig. 7. The growth curve of Saccharomyces cerevisiae in liquid

medium containing 10 mg/l of IAA

The extension period of lag and log. phases is about 96 hrs. which

is greater than both concentrations of lmg/l and 5 mg/l of IAA in

the. liquid medium.

Total cells

Living cells

Time of grwoth in days

0 2 4 6 8 10 12

10

8

6

4

2

0

Log.

no.

of

cells

47

(c) The change in concentration of indole compounds at

different stages of yeast growth

(I) In control culture

During the grovrth stages of yeast, the change of concentration

of exogenous indole compounds in control culture was co-related with

its growth curve. At the beginning of its growth, there was no

indole compounds detected. The increase of indole compounds was

detected after 96 hrs. The examination of the cultures of 72 hrs

growth gave a variety of indole compounds which included IAN and

IAA. The concentration of indole compounds increased graduately

during the stationary phase and reached a.maximum at the death

phase (Fig. 6).

(II) In cultures containing 1 mg/l, 5 mg/l, and 10 mg/1

of IAA

In cultures with external application of IAA, the degradation

of this compound was observed at the early growth periods indicated

by the increase of transmittance. Only when tho degradation of IAA

reached its minimum, the cultures went into stationary phase. In

cultures with 1 mg/l and 5 mg/l of IAA, 96 hrs was required for IAA

degradation. Six days were required for the degradation of IAA in

10 mg/1 culture. After this time the content of indole compounds

increased as in the control culture (Fig. 8).

48

Fig. B. The change of concentration in exogenous indole compounds

at different stages of growth in Saccharomyces cerevisiae

In control culture, the concentration of exogenous indole com-

pounds increases with time and reaches its maximum at death phase.

For those cultures grown in liquid medium containing different

doses of IAA, there is a decrease in indole compound concentration

at first. But it increases after reaching its minimum point. The

minimum point of indole compound concentration indicates the end

of log, phase.

Time of growth in days

0

-1

-5

-10

mg/l. IAA

100

90

80

70

60

50

40

0 2 4 6 8 10 12

%

of

transmttance

49(d) The bioassay of indole compounds

The bioassay are considered to be the most effective method

for determination of the quantity of plant hormones. Avena

curvature test has been tried but failed. The method was recommended

by Sirois (1968). The results in this experiment were unsuccessful

and not in order even for controls. The summarized data are as

follows:

IAA dose

(mg/1)Measurement (mm) Mean (Difference)

0.0

0..1

0.2

0.4

0.8

1.5

3.0

5.0

10.0

7.0

8.8

7.1

9.5

9.5

8.9

13.8

7.5

7,8

6.5

7.0

10.8

10.5

10.1

13.1

10.7

7.0

6.9

6.0

9.0

6.9

8.0

10.1

9.5

8.6

7.1

11.0

8.0

9.0

9.5

11.5

9.1

9.0

11.3

7.5

7.0

9.0

8.5

8.8

10.5

8.3

5.5

9.1

9.8

6.5

8.5

10.5

11.1

9.8

9.8

9.8

13.1

11.7

11.8

7.5

7.5

7.5

8.5

10.5

12.4

9.5

8.9

9.6

6.5

9.1

10.5

11.0

9.8

10.8

10.7

9.5

8.9

7.4

8.7

9.0

9.9

9.7

9.9

10.8

8.6

8.7

(2.5)

(2.3)

(2.5)

(3.4)

(1.2)

(1.3)

According to the; data obtained, they did not indicate any

significance. So maybe the method should be improved.

(1.3)

(1.6)

50

(3) The measurements of gas during the growth stages of yeast

(a) Yield of CO2 per unit time in ordinary fermentation

Using the apparatus described as in previous sections, a

decrease of gas volume was observed during first 15 hrs. Rapid

increase of CO2 yield was observed after 20 hrs of growth, The

yield of CO2 became maximum at 60-80 hrs. A rapid fall of CO2

yield was shown shortly after this time and became very low at

120 hrs (Fig. 9).

If we compared the yield of CO 2 by yeast and its growth

curve, we could find the similarities of these two curves. At

the lag phase of yeast growth, it seemed that oxygen was consumed.

Fermentation began at log. phase and reached to. the highest at late

log. phase and stationary phase. Only a small amount of fermentation

was occurred at the death phase.

(b) Oxygen consumption by yeast during the growth stages.

In the control yeast culture, the oxygen consumption was

observed to be very low from 4-8 hrs. After this period, the

consumption of oxygen became very rapid and reached its maximum

at 24 hrs. Immediately after this period, there was a tremendous

fall in-oxygen consumption (Fig. 10).

51

Fig. 9. Carbon. dioxide yield by Saccharomyces cerevisiae

The negative values indicate that oxygen is used in the early

stages. After this period of oxygen consumption, the culture

enters to fermentation period indicated by large quantity of

receiveCO2 yield. Even though the culture does not any shaking

properand aeration yet the curve obtained still

represents the characteristics of yeastgrowth.

8

7

6

5

4

3

2

1

0

-1

-20 20 40 60 80 100 120 140 160 180 200 240 260 280

Time of growth in hours

Vol.(in

c.c.)

of

CO2

yield/hr.

52

The curve of oxygen consumption for the culture in 1 mg/l

of IAA was basically similar to that of the control culture. Yet

the quantity of oxygen consumed at the maximum point was increased

and there was also a shift of the maximum consumption peak to 32

hrs. The time of high oxygen consumption was also extended (Fig.

10).

In the culture with 5 mg/l of IAA in medium, the curve was

more or less the same as that in the 1 mg/1 culture but with a

greater shift of the point of maximum oxygen consumption to 42 hrs.

It is quite strange at the maximum oxygen point that consumption

of oxygen per unit time was only comparable to that of the control

culture and lower than that of the l-mg/l culture. Yet the quantity

of oxygen consumed as a whole was larger than that of the 1 mg/l

culture (Fig. 10).

In the culture with 10 mg/l of IAA in medium, there was

still a greater shift of the maximum oxygen consumption peak to

44 hrs. and the extension of the higher oxygen consumption period

was longer and higher in quantity of oxygen consumed per unit time

at this point (Fig. 10).

53

Time of growth in hours

Fig. 10. The consumption of oxygen by different types of cultures

of Saccharomyces cerevisiae

Note the extension of time for higher oxygen consumption. It

seems that the higher is the concentration of IAA content per

liter of liquid medium, the longer is the extension period.

Thougn the maximum quantity of oxygen consumption per hour

does not increase proportionally with IAA concentration, yet

total oxygen consumed increases with the concentration of IAA

dose.

01

5

10

mg/l IAA

1100

1000

900

800

700

600

500

400

300

200

100

00 20 40 60 80 100 120 140

mm3

of

O2

consumed

54

(4) The sugar used during the growth stages of yeast

The sugar (glucose) used by yeast in control growth was

quite even during the first 8 days. After this time, the consumption

of sugar was declined to a very low rate (Fig. 11).

The consumptions of sugar by the cultures with 1 mg/1 and

5 mg/l of IAA were very similar. During the first two days, the

consumption of sugar was not very large with only about 10% of the

sugar consumed. The rapid consumption of sugar began from the

third day till the fourth day. Within these two days, about 40,10

of sugar in the medium was utilized. The rapid fall in the rate

of sugar consumption was observed .after the fourth day. There was

less than 1D% of sugar used up in the remaining 8 days (Fig. li).

In the culture of 10 mg/1 of IAA, the sugar consumption in

the first 2 days was' the lowest among the four types of cultures.

There was only about 91/0 of sugar used up. Yet higher sugar

consumption to about 25% was observed from the third day till the

fourth day. Within these two days, about 29% of sugar was'used.

This was not the only time of high sugar consumption during the

growth stages of yeast culture. As indicated in the curve,

actually about 4% of sugar was used up from the third day till

the eighth day. After this high sugar consumption period, the

consumption rate of sugar fell (Fig. 11)d

55

Fig. 11. Percentage of sugar (glucose) remained in solution medium

The sugar used in first two days seems to be less in higher IAA

content of the cultures. Decrease of sugar content in liquid

medium of control culture and culture with 10 mg/l of IAA is

approximately linear with time. In cultures containing 1 and

5 mg/1 of IAA, the decrease of sugar content is very rapid from

second till fourth day.

Time of growth in days

0

1

5

10

mg/L IAA

100

90

80

70

60

50

40

0 2 4 6 8 10 12 14

%

of

sugar

remained

56

(5) The alcohol production at different stages of yeast groith

The production of alcohol (ethanol) in our strain k'ihcn

compared with the wine making strains is rather low. At the

first tvio days, only about 0.11 g per 10 ml of alcohol. v:as

produced and the efficiency of fermentation was 69.2;; according

to Pasteur's percentage. But the production of alcohol increased

readily so as the percentage of efficiency. The final alcohol

production found was 0.71 g per 10 ml of the medium and the efficiency

was increased' to 88.1% (Table 2).

In the cultures containing 1 mg/l and 5 mg/l of IAA, the

quantity of alcohol produced at the first two days made little

difference, if any*., in compare with the control culture. After

the first two days, the quantity of alcohol produced was much

greater than that in control culture. About 0.57 and 0.52 g per

10 ml of liquid medium was found in 1 mg/1 and 5 mg/1 cultures,

respectively. The fermentation efficiency increased significantly

in the culture with 1 mg/l of IAA. About 4,, 5`/0 of fermentation

efficiency was noted at this time. There was an increase in the

final alcohol production and an increase of approximately loo of

fermentation efficiency (Table 2).

57

Table 2 Quantity of alcohol yield during yeast growth and the% by weight of production

,QuantityT I M E 0 F G R 0 W T H I N D A Y S

0 2 4 6 8 10 12Culture's

0 0.11 0.19 0.34 0.56 O64' 0.71*

0 0% 66.0% 70.2% 74.1% 7705% 79.6% 83.5%

0% 69.2% 74.1% 78.2%- 81.8% 83.6% 88.1%**

0 0.12 0.57 0058 0.62 0.68 0.74

10% 71.0% 74.5% 75.6%. 78.4% 80.2% 84.3%

0% 75.1% 78.6% 79.8% 82.8% 84..6% 89.0%

0 0.10 0.52 0.58 0.62 0.68 0.73

5 0% 66.1% 70.4% 73.6% 77.2% 79o8% 84.00

0% 69.8% 74o3% 77.7% 81.5% 84.2% 88.7%

0 0.05 0.28 0.35 0.56 0.64 0.72

100% 60.7% 70.6% 73.3% 76.4% 80.8% 85.1%

0% 64.1% 74.5% 77.4% 80.7% 85.3% 89.8%

Notes* no. in grams/ 10mi** Pasteur's %

53

In culture containing 10 m1/1 of IAA, the quantity of alcohol

produced in the first two days was very low. Only about 0.05 g

per 10 ml of liquid medium was produced and the percentage of

fermentation was 64.1%. The alcohol production and the efficiency

of fermentation increased readily. An increase of 1.'7% of

fermentation efficiency was found at last (Table .2).

59

(6) Other changes during the growth stages of yeast

(a) The cell size

There was no significant change in cell size no matter

with or without the application of IAA in the liquid medium.

The cell size remained quite constant at any growth period. They

ranged from 2.25- 6.00 X 3.00- 7.50 u which 's within Lodder

and Kreger-van Rij's (1952) range from 3.00- 10.00 X 4.50 -15.00

u (Table 3).

(b) Smelling

Flavor is one of the very important factor for evaluation

of wine. The flavor, most probably the smelling of acetyl ester,

increased with the time of fermentation in control culture. In the

cultures containing 1 mg/l and 5 mg/l of IAA, the flavor did not

change within the first 10 days. Yet smelling of mold was noted

at the 12th day. In the culture containing 10 mg/l of IAA, there

was no change in the smelling of flavor even after the 12th day

(Table 4).

(c) Coloring

The color of the. control culture remained creamy white nearly

all the time and changed into creamy yellow at very late stage. In

.culture containing 1 mg/l of IAA, the color of the culture resembled

to that of the control culture in the first few days. It began to

change into creamy yellow after the fourth day: and graduately

60Table 3: Cell size of yeast at different time of growth.

The figures given are. in u

TIME OF GROWTH IN HOURSCultures

0 48 96 144 192 240 288

3.755025 3.75'5.25 3000-.4050 3000-5.25 3000-4.50 3.005.25 2.25-4a5O

o x x x x x x x

6.007.50 6.007.50 4.50-7.50 4050-7.50 5,25-7.50 4.5o-6.00 4.50-6075

3.75-5.25 3.75--4.50 3.00--4.50 3000--4050. 3,00--5025 3.00--6.00 3.75--5.25

1 x x x x x x x

3 0 00--7.50 6 0 00--7.5o 4 0 50--6.00 4 0 50-6.00 3.75-6o75 3 0 7 57.50 4.50--7.50

3.75-5.25 3.75-5.25 3.005.25 30004.50 .3.75-6.00 3.756.00 2o25s5.25

5 x x x x x x x

300'.7.50 5.257.50 4.50-6.75 3.00-6.oo 5.257.50 30*75=7.50 40506.00

3.75--5.25 3.00--4.50 3.00--4.50 3.00-4.50 3.00-4.50 3.00-5.25 3.00-4050

10 x X. x x x x x

3.00--7.50 4.50-6.00 3075-6.75 300o--6.75 4.506.75 5.25-7.50 3.00-6.00

61Table 4. Summary of data available at present time

62developed a green shade color like the sugar-cane juice. I n the

culture containing 5 mg/l of IAA, the color was also resembled to

the control in the first few days. The development of the green

shade sugar-cane juice`color was also observed at the later stages.

In culture containing 10 mg/l of IAA, the color remained white at

the very early stages and changed to pinkish color at the later

stages (Table 4).

The cell color in mass of control culture remained creamy

all the time observed. But the color of.cells of the cultures

containing 1 mg/1 of IAA changed to dirty grey at later stages.

The cells of 5 mg/l dosage colored dirty grey at the later stages

and development of pink color .ryas also observed at later stages.

In culture containing 10 mg/l of IAA, the color of cells was pink

and the color intensity increased with time.

(d) Budding

Budding seemed increased with age of the culture, and the

number of cells in each budding group increased with time. The

cells within one budding group was not all alive by ....this staining

method. Sometimes empty cells were observed in a budding group

together with some living cells.

63Discussion

(1) Indole compounds production

The production of indole compounds and other plant hormones

is no longer new to the biologists. Since Gruen 's review (1959a)

on auxins in fungi, we know that some of the fungi belong to the

four divisions of the broad classification produce one or more kinds

of auxins or plant hormones. Since higher plants produce auxins and

other plant hormones with some in resemblance to that in fungi and

others quite different. There probably is an indication of

evolutionary line in.the plant kingdom between fungi and higher plants.

However, even with different fungi there is a difference in

indole compounds production. Besides the common indole compound of

IAA produced in this experiment, Aspergillus niger NAC2 produces

indole, trytophan, and skatole but Saccharomyces cerevisiac produces

indoleacetonitrile and indolealdehyde. This may indicate that

different fungus has its own way in indole metabolism.

The production of auxins is not common in genus Aspergillus

(Curtis, 1953). In this experiment both Asnergillus nicer and A. nicrer

NAC2 give positive results on spot test of IAA. Negative results

are obtained on A. flavus, A. t,wentii, L. fumigatus, L. or zac and

a few other species. As a group this might indicate that A. n_iger

is different from other species of Asper cgillus.

64

(2) Physiology of auxin on yeast

In general 'auxin showed some inhibitory effect on fungi

(Gruen, 1959a). However Turfitt (1941) found that IAA, o(-NAA, and

(3-NAA (n.l- 1.0 mg/l) promoted growth of Saccharomyces cerevisiae

in a synthetic medium, Auxin has also an effect of cell expansion

in yeast (Yanagishima et all 1966-70).

In this experiment, auxin inhibition on growth of S. cerevisiae

was noted too. But no cell expansion was observed. As reported by

Yanagishima (1970), the effect of auxin on yeast to induce cell

expansion only occurred in those strains which are homothallic.

Since we cannot induce ascus formation by culturing on calcium

sulfate blocks, it is assumed that the strain used in this experiment

is heterothallic and hence no cell expansion is essential.

The increase in indole concentration during the growth

period of yeast indicates that the indole compounds have some

re-etion to the --senescence. There is evidence that the concentration

of indole compounds is the highest at death phase. IAA seems

responsible for the inhibition of growth or the extension of early

growth stages but it has nothing to do with r.,,enescence. If it is

so, it-must-has-the effect of prolonging senescence time. Professor

Bellamy in a public lecture given on December 15, 1971 at the

University of Hong Kong on subject of Current Ideas in Gerontology

65stated that" ....... the extension of prematurational stages would

result in the extension of maturational and post maturational stages.

After the maturational stage is reached' there would be no hope of

rejuvenation. All the best is to stop or to remain at that stage

of growth. The extension of early stages in yeast growth by the

external application of IAA in this trial gives also some evidence

to this statement.

The large consumption of oxygen at early stages indicates that

the metabolism of yeast cell is induced by application of IAA. IAA

inhibits the increase in cell population but it does not inhibit the

metabolism of yeast.-. Sarkissian (1965) found that IAA leads to

elevation of activity of citrate' sinthase from corn scutella in vitro.

He proposed that IAA regulates an enzyme by transmitting a biochemical

signal to the protein via SH on the protein molecule and that

probably in all other instances of metabolic or structural regulation

IAA acts in the same manner of transmitting its signal to an SH group.

If this is true, it should be reasonable that the yeast cultures

with the application of IAA have higher metabolical rate. This

experiment gave positive results as demonstrated by the higher oxygen

consumption and higher percentage of alcohol production.

The dosage effect of IAA on the growth of yeast is not in

good proportion. At low concentration as 1 mg/1 of IAA, it is

effective only at the early stages. In 5 mg/1 of IAA, it behaves

somewhat like that of 1 mg/1 in*general but sometimes it differs

and is inclined to behave like that in 10 mg/l. This may be the

66transitional dosage that gives the first step to affect the growth

of yeast. In 10 mg/l of IAA, the culture behaves quite orderly

and shows typical characters of IAA action. But no matter what

quantity of IAA dosage is used, it is intented to be broken down

to a tolerable concentration before the culture can get into the

further growth stages.

The sugar used up by yeast cultures was greatest in the

early stages at 1 mg/1 and 5 mg/l of IAA. The penetration of sugar

through the yeast at this dosages might be promoted, hence more

molecules of sugar per unit time may get into the yeast cells and

be broken down by the enzymes inside.

The red pigment that produced in the cultures containing

5 mg/l and 10 mg/1 of IAA is expected. Reddening of yeast cells

by culturing conditions has been reported (Cutts and Rainbow, 1950;

Chamberlain et al, 1952). It is noted that even yeast grown in 9%

glucose may induce production of red pigment.

67

(3) Suggestions for application

Yeast has been well known for its production of alcohol,

glycerol and other important substances through fermentation. Our

data indicate that with the application of IAA, both the production

of alcohol and the percentage yield are higher. Time and raw

materials such as sugar are also saved thereby increasing the basic.

value of its yse in brewing industries. The application of IAA in

yeast cultures while-improving the production of ethanol may probably

be extended to other processes of fermentation industry. Further

investigations are needed for elucidating the value of IAA application.

The fact that flavor is not spoiled by the application of

IAA to the fermentation medium which makes it probable that the

same procedure could apply to wine making industries. Further

investigations on this point may even lead to production of special

flavor in wines.. If not, at least an enrichment-of flavor may

successfully be induced since it was experimentally shown that at

the end of the growth period, the flavor in 10 mg/1 of IAA seemed

to be sweeter and more concentrated.

68Summary

Investigations of production of indole compounds by Aspergillus

ni er NAC2 and Saccharomyces cerevisiae have been made on fungal

materials and culture media with paper chromatography. Identification

of the indole compounds has been made according to the Rf values in

the iso-propanol, ammonia, and water solvent; and the color reaction

with Ehrlich, Salkowski, diazotized nitroaniline and diazotized

sulfanilic acid reagents. Methods ysed in the investigation were

very varied. Therefore each mothod was stated separately and discussed

whenever it was needed.

Indole, indoleacetic acid, and tryptophan have been found in

the culture medium of Aspergillus; and skatole and two unidentified

indole compounds have been observed in mycelial mats. Indoleacetonitrile

and indoleacetic acid have been found in culture medium of Saccharomyces

cerevisiae; and indoleacetonitrile, indolealdehyde, indoleacetic acid,

and three other indole compounds have been observed in yeast cells.

The exogenous indole compounds have been studied in relation

to time and concentration. It is found that the concentration of

indo.1e compounds increases with time and reaches its maximum at

death phase of the culture.

The external application of IAA to the yeast culture extends

69

the early growth stages. The cultures do not enter stationary phase

until the exogenous IAA has been degraded.

The applicat,on of IAA promotes the consumption of oxygen

and also the efficiency of fermentation. No expansion of the cells

has been found. IAA does not break down the flavor during fermentation

but intensifies it at higher dosage.

Failure in bioassay of IAA in the method described by Sirios

has also been encountered in this experiment. A method of distingushing

the dead cells from living has been developed.

70

吲 □ 激 素 之 產 生 及 其 對 菌 類 各 生 長 期 之 影 響

黑 麯 菌 及 酵 母 菌 所 產 生 之 吲 □ 化 合 物 ， 可 用 紙 上 色 層

分 析 法 檢 定 。 以 異 丙 醇 、 氨 及 水 作 溶 劑 ， 求 出 其Rf

值 ， 再

用 四 種 顏 色 反 應 確 定 其 類 別 。

于 黑 麯 菌 之 培 養 基 中 ， 分 離 出 吲 □ ， 吲 □ 乙 酸 及 色 氨

酸 ； 于 其 菌 體 內 則 分 離 出 臭 糞 素 及 兩 種 未 鑑 定 之 吲 □ 化 合

物 。 于 酵 母 菌 之 培 養 基 中 ， 分 離 出 吲 □ 乙 腈 及 吲 □ 乙 酸 ；

于 其 菌 體 內 則 分 離 出 吲 □ 乙 腈 ， 吲 □ 乙 酸 ， 吲 □ 醛 及 三 種

其 他 之 吲 □ 化 合 物 。

于 菌 類 之 生 長 中 ， 培 養 基 內 吲 □ 化 合 物 累 增 ， 當 生 長

進 入 死 亡 期 時 則 增 至 項 點 。 外 加 之 吲 □ 乙 酸 ， 能 延 長 酵 母

菌 生 長 之 前 期 。 直 至 外 加 之 吲 □ 乙 酸 被 分 解 ， 以 後 酵 母 菌

生 長 才 進 入 穩 定 期 。

吲 □ 乙 酸 能 促 進 酵 母 菌 對 氧 之 吸 取 及 其 發 酵 效 率 ， 並

不 破 壞 其 香 味 ， 且 于 較 高 濃 度 時 有 增 進 其 香 味 之 作 用 。 然

此 實 驗 並 未 發 現 細 胞 有 膨 大 作 用 。

薩 利 澳 氏 所 描 述 對 吲 □ 乙 酸 之 生 物 測 定 法 ， 在 本 實 驗

之 效 果 未 達 理 想 。 至 於 酵 母 菌 細 胞 生 死 檢 定 法 ， 本 實 驗 則

建 議 一 種 簡 新 方 法 。

71Acknowledgement

I heartily thank Dr. Yun-shen Bau for being my super-

visor and helping me a lot during the past two years. I am

indebted to Dr. Shu-ting Chang for being my advisor and giving

me valuable advice whenever needed. It is also my wish to

thank Professor Harry Wang, Professor Peter K.AChen and Dr.

James Ma for their reviewing and criticizing this thesis.

To all my friends and colleagues at this institute I

wish to express my thanks for their kind co-operation and help.

I shall never forget Mr. S. F. Lee for his help in preparing

slides and lessening my washing burden.

Finally I am very grateful to this University for the

financial assistance and all the convenience rendered me.

72Literature cited

Abeles, F. B. and L. E. Forrence. 1970. Temporal and hormonal

control of p-1,3-glucanase in Phaseolus vulgaris L.. Plant

Physiol. 45:359-400.

Ames, B. N. and H. K. Mitchell. 1952. Phenols, aromatic acids,

and indole compounds. In: A manual of paper chromatography and

paper electrophoresis. Block, R. J., E. L. Durrum and G. Zweig,

eds. Academic Press, N. Y. p305.

Bassham, J. and M. Calvin. 1957. The role of carbon in photosyn-

thesis. Prentice-Hall, Englewood Cliffs, N. J.

Batista, A. C., C. S. Fernandes and E. A. de Luna. 1966. Investi-

gations on fungi from soils producing hormones and other substances

promoting or stimulating-plant growth. In: Proceedings of the

institute of mycology of the Federal University of Pernambuco at

the 17th National Congress of the Botanical Society of Brazil,

Brasilia, 17-25 January, 1966. Univ. Fed. Pernambuco Inst. Micol.

Atas. 1:424-433.

Beadle, G. W. and E. Z. Tatum. 1941. Genetic control of biochemical

reactions in Neurospora. Proc. Natl. Acad. Sci. U. S. 27:499-506.

Bentley, J. A. 1950. An examination of a method of auxin assay using

the growth of isolated sections of Avena coleoptiles in test solu-

tions. J. Exp. Bot. 1:201-213.

Boysen- Jensen, P. 1919. Transmission of the phototropic stimulus

in the coleoptile of the oat seedling. Berichte d. Deutsche

Botanische Gesellschaft 28:118-120.

73Bray, H. G. 1950. Phenols, aromatic acids, and indole compounds.

In: A manual of paper chromatography and paper electrophoresis.

Block, R. J., E. L. Durrum and G. Zweig, eds. Academic Press,

N. Y. p305.

Chandramohan, D. and A. Mahadevan. 1968. Epiphytic micro-organisms

and IAA synthesis. Planta 81:201-205.

Chang, K. H. 1964. Physiology of bacteria. People's Health

Publications, Peking.

Cherry, J. H. 1960. Regulation of invertase in washed sugar beet

tissue. In: Biochemistry and physiology of plant growth substances.

Wightman, F. and G. Setterfield, eds. Runge Press, Ottawa. p417-431

Cleland, R. E. 1968. Wall extensibility and the mechanism of auxin-

induced cell elongation. In: Biochemistry and physiology of plant

growth substances. Wightman, F. and G. Setterfield, eds. Runge

Press, Ottawa. p613-624-

Curtis, R. W. 1958. Curvatures and malformations in bean plants

caused by culture filtrate of Aspergillus niger. Plant Physiol.

32:263-269.

Darwin, C. and F. Darwin. 1860. Sensitiveness of plants to light:

its transmitted effects. The power of movement in plants. London.

Dox, A. We 1910. Cultivation. In: The genus Aspergillus. Raper,

K. Be and D. I. Fennell, eds. Williams and Wilkins, Baltimore. p36.

Dvornikova, T. P., G. K. Skryabin and N. N. Suvorov. 1968. Trans-

formation of tryptamine by Aspergillus niger. Mikrobiologiya

37:228-232.

74Dvornikova, T. P., N. N. Suvorov and G. K. Skryabin. 1968. Hydro-

xylation in fifth position of Indole-3-acetic acid by a submerged

culture of Aspergillus niger, %likrobiologiya 37:44-47.

Epstein, E. and P. Miles. 1967. Identification of indole-3=-acetic

acid in the Basidomycete Schizophyllum commune. Plant Physiol.

42:911-914.

Fenner, L. M. and L. R. Fate. 1947. Ceratostomella ulmi on elm

bark treated with 2,4-dichlorophenoxy acetic acid. Phytopath.

37:925-923.

Galston, A. VI., S. Lavee and B.. Z. Siegel. 1966. The induction

and repression of peroxidase isozymes by 3-indoleacetic acid.

In: Biochemistry and physiology of plant growth substances.

Wightman, F. and G. Setterfield, eds. Runge Press, Ottawa.

p455-472.

Galston, A. W. and W. K. Purves. 1960. The mechanism of action

of auxin. Ann. Rev. Plant Physiol. 11:239-276.

Glasziou, K. T., K. R. Gayler and J. C. Waldron. 1960. Effects

of auxin and gibberellic acid on the regulation of enzyme

synthesis in sugar-cane stem tissue. In: Biochemistry and

physiology of plant growth substances. Wightman, F. and G.

Setterfield, eds. Runge Press, Ottawa. p433-442.

Goldsmith, M. H. M. 1966. Movement of indoleacetic acid in

coleoptiles of Avena sativa L. II. Suspension of polarity

by total inhibition of the basipetal transport. Plant Physiol.

41:15-17.

75Goldsmith, M. H. M. 1967. Movement of.pulses of labeled auxin

in corn coleoptiles. Plant Physiol. 42:253-263.

Gordon, S. A. and R. P. Weber. 1951. Colorimetric estimation

of indoleacetic acid. Plant Physiol. 23:192-195.

Gruen, H. E. 1959a. Auxins and fungi. Ann. Rev. Plant Physiol.

10:405-440.

Gruen, H. E. 1959b. Growth and development of isolated Phycomyces

sporangiophores. Plant Physiol. 34:158-168.

Gruen, H. E. 1965. The production of indoleacetic acid by

Phycom ces blakesleeanus. Mycologia 57:683-695.

Haber, A. H., D. E. Foard, and S. W. Perdue. 1969. Actions of

gibborellic and abscisic acids on lettuce seed germination

without actions on nuclear DNA synthesis. Plant Physiol.

44:463-467.

Hall, M. A. and L. Ordin. 1963. Auxin-induced control of cellulose

synthetase activity in Avena coleoptile sections. In: Biochemistry

and physiology of plant growth substances. Wightman, F. and

G. Satterfield, eds. Runge Press, Ottawa. p659-G71.

Hessayon, D. G. 1952. Effect of auxins on the mycelial growth

of Fusarium oxysporum var. cubense. Nature (London) 169:

003-304.

Hocking, D. 1967. Zygospore initiation, development and

germination in Phycomyces blakesleeanus. Trans. Brit. Mycol.

Soc. 50:207-220.

76Holm, R. E., T. J. O'Brien, J. L. Key and J. H. Cherry. 1970.

The influence of auxin and ethylene on chromatin-directed

ribonucleic acid synthesis in soybean hypocotyl. Plant Physiol.

45:41-45.

Hopkins, F. G. and S. W. Cole. 1903. Degradation of amino acid.

In: The life of bacteria, their growth, metabolism and relation-

ships. Thimann, K. V.,ed. Macmillan, N. Y. 1955.

Jacobsen, D. W. and C. H. Wang. 1966. The biogenesis of ethylene

in Penicillium digitatum. Plant Physiol. 43:1959-1966.

Jepson, J. B. 1960. Indoles and related Ehrlich reactors of

mainly medical interest. In: Chromatographic and electrophoretic

techniques. Vol. I. Smith, I., ed. Heinemann, London. p183-211.

Jerebzoff-Quintin, S. 1967. On the subject of the antiauxin

effects of phthalic acid and some of its esters in Nectria

galligena Bres. R. Hebd. Seances. Acad. Sci. Ser. D. Sci.

Natur. Paris 264:1043-1046.

Khan, A. A. 1968. Inhibition of gibberellic acid induced germina-

tion by abscisic acid and reversal by cytokinins. Plant

Physiol. 44:1463-1465.

Kinoshita, K. 1927. The kojic acid fermentation. In: Industrial

microbiology. Prescott, S. C. and C. G.Dunn, eds. McGraw-Hill,

N. Y. p610.

77Knaysi, G. 1935. A microscopic method of distingushing dead

from living bacterial cells. J. Bact. 30:193-206.

Leifson, E. 1951. Staining methods. In: Laboratory methods

in microbiology. Harrigan, W. F. and M. E. McCance, eds.

Academic Press, N. Y.

Letham, D. S. 1967. Chemistry and physiology of kinetin-like

compounds. Ann. Rev. Plant Physiol. 18:349-364.

Letham, D. S. 1969. Cytokinins and their relation to other

phytohormones. Bioscience 19:309-317.

Libbert, E., S. Wichner, E. Duerst, R. Kunert, W. Kaiser, A.

Manicki, R. Nlateuffel, E. Riecke and R. Schroder. 1968.

Auxin content and auxin synthesis in sterile and non-sterile

plants, with special regard to the influence of epiphytic

bacteria. In: Biochemistry and physiology of plant growth

substances. VVightman, F. and G. Setterfield, eds. Runge

Press, Ottawa. p213-230.

Lodder, J. and N. J. W. Kreger-van Rij. 1952. The yeast. In:

Industrial microbiology. Prescott, S. G. and C. G. Dunn, eds.

McGraw-Hill, N. Y. p16.

Lodhi, F., M. V. Bradley and J. C. Crane. 1968. Auxins and

gibberellin-like substances in parthenocarpic and non-

parthenocarpic syconia of Ficus carica L., CV King. Plant

Physiol. 44:555-561.

78Lyon, C. J. 1965a. Auxin transport in geotropic curvatures of

a branched plant. Plant Physiol. 40:18-24.

Lyon, C. J. 1965b. Action of gravity on basipetal transport

of auxin. Plant Physiol. 40:953-961.

MacLachlan, G. A., E. Davies and D. F. Fan. 1968. Induction of

cellulase by 3-indoleacetic acid. In: Biochemistry and physiology

of plant growth substances. Wightman, F. and G. Setterfield,

eds. Runge Press, Ottawa. p443-453.

Masingale, R. E., J. E. Lewis, S. R. Bryant and C. S. Skinner.

1968. Inhibition of dichlorophenoxyacetones on auxin-induced

growth of Avena coleoptile sections. Plant Physiol. 44:641-

MaCready, C. C. 1966. Translocation of growth regulators. Ann.

Rev. Plant Physiol. 17:283-294.

McMorris, T. C. 1967. Chemistry of antheriodiol hormone-A

a sexhormone of the water mold Achlya bisexualis. Science

156:3774.

Miller, C. 0., F. Skoog, F. S. Okumura, M. H. von Salza, and F. M.

Strong. 1955. Structure and synthesis of kinetin. J. Am. Chem.

Soc.. 77:2662.

Moore, D. J. and W. R. Eisinger. 1968. Cell wall extensibility:

Its control by auxin and relationship to cell elongation. In:

Biochemistry and physiology of plant growth substances. Wightman,

F. and G. Setterfield, eds. Runge Press, Ottawa. p625-645.

644.

79Naqvi, S. M., R. R. Dedolph and S. A. Gordon. 1965. Auxin

transport and geoelectric potential in corn coleoptile sections.

Plant Physiol. 40:966-968.

Naqvi, S. M. and S. A. Gordon. 1965. Auxin transport in flowering

and vegetative shoots of Coleus blumei Benth. Plant Physiol.

40:116-118.

Naqvi, S. M. and S. A. Gordon. 1966. Auxin transport in Zea mays

L. coleoptiles I. influence of gravity on the transport of

indoleacetic acid-2 14 C. Plant Physiol. 41:1113-1118.

Naqvi, S. M. and S. A. Gordon. 1967. Auxin transport in Zea mays

coleoptiles II. influence of light on the transport of

indoleacetic acid-2 14C. Plant Physiol. 42:138-143.

Norberg, Sven-Olov. 1958. Studies in the production of auxins

and other growth stimulating substances by Exobasidium. Symb.

Bot. Upsal. 19:1-117.

Palmer, J. M. 1968. The effect of some plant growth substances

on the induction of enzymatic activities in thin slices of

plant tubers. In: Biochemistry and physiology of plant growth

substances. Wightman, F. and G. Setterfield, eds. Runge Press,

Ottawa. p401-415.

Peng, W. T. 1964. Experiments in Micro-biochemistry. Scientific

and Technological Publications, Shanghai.

Penner, D., J. E. DeVay and P. Sackman. 1969. The influence of

syringomycin on ribonucleic acid synthesis. Plant Physiol.

44:806-808.

80Phelps, R. H. and L. Sequeira. 1960. Auxin biosynthesis in a

host-parasite complex. In: Biochemistry and physiology of

plant growth substances. V`Jightman, F. and G. Setterfield, eds.

Runge Press, Ottawa. p197-212.

Pohl, H. P. and A. I. Hawk. 1966. Separation of living and

dead cells by dielectrophoresis. Science 152:647-649.

Potter, M. C. 1911. Redox potential. In: Methods in microbiology.

Norris, J. R. and D. W. Ribbons, eds. Vol. II. Academic Press,

N. Y. p92-93.

Prochazka, Z.,V. Sanda, and K. Macek. 1960. Methods for the

detection of biochemical compounds on paper and thin layer

chromatograms, with some notes\on separation. In: Data for

biochemical research. Davison, R. M. C., D. C. Elliot, W. H.

Elliot and K. M. Jones, eds. Oxford Univ. Press. p551.

Rabotnova, I. L. 1963. The effects of physical and chemical

conditions (pH and rl-12) in microbial metabolisms. Chinese

translation by the Scientific Publications, Peking.

Raper, J. R. 1951. Sexual hormones in Ac. Am. Sci.

39:110-120.

Ray, P. M. and A. A. Abdul-Baki. 1968. Regulation of cell wall

synthesis in response to auxin. In: Biochemistry and physiology

of plant growth substances. Wightman, F. and G. Setterfield, eds.

Runge Press, Ottawa. p647-658.

81Runkova, L. V. and V. V. Mazin. 1960. Effect of Plasmodiphora

brnssicae Wor. infection on the concentration and nature of

indole derivatives in Brassica roots. Fiziol. Rast. 15:778-

784.

Salkowski, 1830. Degradation of amino acid. In: The life of

bacteria, their growth, metabolism and relationships. Thimann,

K. V., ed. Macmillan, N. Y. 1955.

Sarkissian, I. V. 1968. Nature of molecular action of 3-indole-

acetic acid. In: Biochemistry and physiology of plant growth

substances. Wightman, F. and G. Setterfield, eds. Runge Press,

Ottawa. p473-485.

Gen, S. P. and A. C. Leopold. 1954. Paper chromatography of

plant growth regulators and allied compounds. Physiol.

Planta. 7:98-103.

Shih, C. Y. and L. Rappaport. 1970. Regulation of bud rest in

tuber of potato, Solanum tuberosum L. Plant Physiol. 45:

33-36.

Sirois, J. C. 1968. A quantitative coleoptile elongation test

for growth regulators. In: Biochemistry and physiology of

plant growth substances. Wightman, F. and G. Setterfield,

eds. Runge Press, Ottawa. p1611-1618.

Stowe, B. B. and K. V. Thimann. 1954. The paper chromatography

of indole compounds and some indole-containing auxins of plant

tissues. Arch. Biochem. Biophys. 51:499-516.

82Stowe, B. B., M. Vondrell and E. Epstein. 1968. Separation and

identification of indoles of maize and vioad. In: Biochemistry

and physiology of plant growth substances. Wightman, F. and G.

Sotterfield, eds. Runge Press, Ottawa. p173-102.

Tom, C. F., S. K. Ncr and Y. S. Bau. 1968. An annotated checklist

of Hong Kong fungi I. N.A.C. Acad. Ann. 10:97-124.

Thimann, K. V. 1963. Plant growth substances; past, present

and future. Ann. Rev. Plant Physiol. 14:1-18.

Thumberg, T. 1920. Redox potential. In: Methods in microbiology.

Vol. II. Norris, J. R. and D. IN. Ribbons, eds. Academic Press,

N. Y. p97.

Turfitt, G. E. 1941. The effects of auxin on fungi. In: Auxin

and fungi. Gruen, H. E., ed. Ann. Rev. Plant Physiol. 10:

405-440.

Uden, N. van and A. Madeira-Lopes. 1970. Kinetics and energetics

of yeast growth. In: the yeasts. Vol. II. Rose, A. H. and J.

S. Harrison, eds. Academic Press, London. p97.

Walton, D. C., G. S. Soofi and E. Sondheimer. 1970. The effects

of abscisic acid on growth and nucleic acid synthesis in

excised embryonic bean axes. Plant Physiol. 45:37-40.

Went, F. W. 1926. On cgrov.wth-accelerating substances in the

coleoptile of Avena sativa. Proc. Kon. Akad. Weten.,

Amsterdam. 30:10-19.

83Wightman, F. and G. S etterfield, eds. 1968. Biochemistry and

physiology of plant growth substances. Runge Press, Ottawa.

Vitham, F.. H. 1968. Effects of 2,4-dichlorophenoxyacetic acid on

the cytokinin requirement of soybean cotyledon and tobacco stem

pith callus tissues. Plant Physiol. 43:1155-1157.

Volf, F. T. 1952. Production of indole acetic acid by Ustilago

zone and its possible significance in tumor formation. Phytopath.

42:147-149.

Yabuta, T. and T. Hayasi. 1930. Gibberell'ic acid and the

gibberellins. In: Industrial.microbiology. Prescott, S. C.

and C. G. Dunn, eds. McGraw-Hill, N. Y. p619-G42.

Yanagishima, N. and C. Shimoda. 1960. Auxin-induced expansion

growth of cells and protoplasts`of yeast. Physiol. Plant.

21:1122-1120.

Yanagishima, N., C. Shimoda,T..Takahashi and N. Takao. 1970.

Responsiveness of yeast cells to auxin, animal sex hormones

and yeast sexual hormones in relation to.sex controlling genes.

Develop. Growth Differ. 11:277-206.